Motoki Yonekawa

Clinical study of therapeutic angiogenesis by autologous peripheral blood stem cell (PBSC) transplantation in 92 patients with critically ischemic limbs

Patients with critically ischemic limbs due to maintenance hemodialysis and diabetes are increasing in number markedly in Japan. The difficulty of treating critically ischemic limbs is well recognized. Despite active medication and surgical therapy, many critically ischemic limbs are amputated. Ninety-two patients with critically ischemic limbs were treated by transplantation of autologous peripheral blood stem cells (PBSCs). The stem cells were mobilized into the peripheral blood by administration of granulocyte colony stimulating factor (G-CSF). The mobilized mononuclear cells were separated by an apheresis technique using a centrifuge. The separated mononuclear cells contained approximately 4.0 × 107 CD34-positive cells. The collected cell suspension was divided into aliquots of 0.5–1.0 ml and transplanted into the muscle of ischemic limbs at 50–70 transplantation points. At 1.5 months after PBSC transplantation, a strong immunostaining of CD34-positive cells and factor VIII, as well as capillary formation, was observed in the muscles into which stems cells had been transplanted. In each patient tested, the serum vascular endothelial growth factor (VEGF) level increased after stem cell transplantation; the mean VEGF level increased by 176%. Of 11 diabetic patients (DM) who were not receiving hemodialysis (HD), there were no amputees regardless of their Fontaine classification. Of 19 patients in the HD(+)DM(−) category, there were no amputations in Fontaine stage I, II, and III patients, whereas three limbs and one toe were amputated in Fontaine stage IV patients. Of 13 patients in the HD(−)DM(+) category, none of the Fontaine stage I, II, or III patients underwent amputation, but six Fontaine stage IV patients underwent amputation. Of 49 patients in the HD(+)DM(+) category, 38 (78%) were classified as Fontaine stage IV, 71% (27/38) of whom had a toe or a limb amputated. In nine patients over 80 years of age, one toe and one limb were amputated. Nondiabetic, nondialyzed patients with ischemic limbs are strongly indicated for stem cell transplantation regardless of Fontaine classification. Therapeutic angiogenesis is effective for critically ischemic limbs resulting from hemodialysis and diabetes until Fontaine stage III, but is of limited effectiveness for stage IV cases.

Prevention of Limb Amputation in Patients with Limbs Ulcers by Autologous Peripheral Blood Mononuclear Cell Implantation

Abstract:  There are many cases of amputation of ischemic limbs of dialysis patients due to diabetes, despite the availability of medicine therapy and vascular by‐pass operations. As there is extensive ruin of the vascular bed due to diabetes, vascular regeneration therapy by stem cell implantation is effective. Thirty patients with ischemic limbs due to diabetes (not including type‐I) and on dialysis for chronic renal failure (19 cases), diabetes (5 cases), dialysis patients without diabetes (4 cases), and arteriosclerosis obliterans (ASO, 2 cases) were treated by autologous peripheral blood stem cell (PBSC) implantation where imminent amputation was under consideration. Granulocyte Colony Stimulate Factor (G‐CSF: 5 µg/kg/day) was administered subcutaneously for 4 days before PBSC collection, that was carried out using a centrifuge (Spectra and/or CS3000) via the vein. The collected PBSC, containing 4.2 × 107 of CD 34 positive cells, was divided into units of 0.5–1.0 mL and implanted, without any purification, to the ischemic area of the limbs in about 65 points. In 21 cases, normalization of limb temperature was observed by thermograph, and symptoms also improved. The result of this first attempt of PBSC implantation is that we were able to save 22 ischemic limbs. This is the first large report of the application of regenerative medicine to peripheral ischemic limbs.

Adenocarcinoma arising in gastric heterotopic pancreas: Clinicopathological and immunohistochemical study with genetic analysis of a case

Heterotopic pancreas in the stomach is a relatively common congenital condition, but the risk of malignant transformation is extremely low. In this study, we describe a case of adenocarcinoma arising from a gastric heterotopic pancreas and we consider its morphological and immunohistochemical features and genetic analysis, in order to examine its histogenesis. This unusual sequela was seen in a 57‐year‐old woman. Image studies showed a protruding lesion with a central ulcer located in the lesser curvature from the angle to the body of the stomach. A biopsy specimen confirmed this lesion as adenocarcinoma before total gastrectomy. The tumor showed mixed patterns of solid neoplastic‐cell proliferation and moderately differentiated glandular structures, and also showed transitional lesions to obvious malignancy, that is, dysplasia, or adenocarcinoma in situ. Neoplastic cells had positive immunoreactivity for carbohydrate antigen (CA) 19‐9, mucin (MUC) 1, and insulin, and the mutant allele‐specific amplification method revealed a point mutation at K‐ras codon 12 (GGT [Gly]→GAT [Asp]), which is the most common mutational change observed in patients with pancreatic carcinoma. The features of the present case provide clear evidence that this tumor originated from heterotopic pancreatic tissue rather than from gastric epithelium.

Artificial liver support at present and in the future.

Liver failure is a fatal disease. Liver transplantation is the only established treatment for liver failure; however, donor shortages remain problematic. In the United States and Europe, artificial livers as a bridge to liver transplantation are being considered. In Japan, we have taken a different approach to the treatment of end-stage liver diseases because of the characteristics of the health-care insurance system, regulated by the government. Furthermore, cadaveric liver transplantations are unsuited to the social mores of Japanese culture. Practically speaking, we believe that plasma exchange (PE) and continuous hemodiafiltration (CHDF) are the most effective therapies for the treatment of liver failure, although randomized controlled studies are needed to determine their effects. Overall, we believe that the first line of treatment for liver failure should be PE and CHDF, and the second line should be bioartificial liver support. In the near future, we hope that both gene therapy and regenerative medicine will contribute to the development of a functional artificial liver.

Transient increase in telomerase activity of proliferating fibroblasts and endothelial cells in granulation tissue of the human skin

Although granulation tissue formation is an important step for second‐intention wound healing, the molecular events underlying this process are still poorly understood. To investigate the role of telomerase in the formation of granulation tissue, we measured the activity of this enzyme and determined the expression and localization of human telomerase reverse transcriptase mRNA using human skin samples. Telomerase activity in the tip of the granulation tissue where fibroblasts actively proliferate was detected at a level 5.6 ± 1.5 times higher than that at the edge of the tissue when using a polymerase chain reaction‐based telomeric repeat amplification protocol assay coupled with enzyme‐linked immunosorbent assay. This, together with the findings from semiquantitative reverse transcriptase‐polymerase chain reaction and in situ hybridization of human telomerase reverse transcriptase, revealed that proliferating cell nuclear antigen‐positive fibroblasts and endothelial cells in the progressing granulation tissue showed de novo activation of telomerase with high human telomerase reverse transcriptase mRNA expression. This condition may be a prolongation of cellular replicative capacity taking advantage of the positive regulatory dynamics of cell growth. We conclude that the regulation of telomerase activity may play an important role in granulation tissue formation in wound healing.

Successful treatment of ulcerative colitis with leukocytapheresis using non-woven polyester filter

Abstract:  Ulcerative colitis is a chronic inflammatory disease of the rectum and colon. Although the pathogenesis of ulcerative colitis is not fully elucidated, cell‐mediated immunity plays an important role in disease pathogenesis. Leukocytapheresis is a newly emerging therapy to eliminate activated leukocyte from systemic circulation. We have studied the effects of leukocytapheresis on patients with ulcerative colitis who had failed to respond to conventional therapy. A total of 51 patients with ulcerative colitis were treated with apheresis using a non‐woven polyester fiber filter (Finecell, Asahi Medical Co., Tokyo, Japan) originally developed as a microcoagulation elimination filter for massive transfusion. Of the 51 patients, 33 (64.7%) achieved clinical remission manifested by clinical activity and colonoscopic findings without any adverse effects. This result suggested that leukocytapheresis using Finecell might serve as an alternative therapy for ulcerative colitis as other leukocytapheresis using centrifugation or column.

Study of prolonged administration of lanthanum carbonate in dialysis patients.

Data of 36 months were accumulated regarding the effects of lanthanum carbonate (LA) on serum phosphate concentrations in dialysis patients. Fifty‐three patients (average age and dialysis history 58.4 years and 9.1 years) were included in this study who have been receiving outpatient treatment since March 2009, and who have been unable to maintain serum phosphate concentrations of ≤6.0 mg/dL via traditional therapeutic agents used for hyperphosphatemia. Patients were given dosage of LA in addition to, or instead of, co‐hyperphosphatemia treatments already being received. Mean dosages of calcium carbonate (CC) and sevelamer hydrochloride (SH) before starting LA administration were 1301.9 mg and 2462.3 mg, respectively. Dosage of LA for all cases was 750 mg at initial dose; 1528.3 mg at 5 months; and 1416.7 mg at 30 months. Dosage of other phosphate binders were 905.7 mg of CC and 820.8 mg of SH at 5 months; and 687.5 mg of CC and 1031.3 mg of SH at 30 months. Serum phosphorus levels (P levels) were significantly decreased at 1 month of LA administration, and continued until 30 months of La treatment. These results suggest that LA successfully controlled serum P and Ca concentrations simultaneously within target ranges without affecting serum intact parathyroid hormone concentration, although further long‐term prospective cohort study on LA would be required.

A Case Report of the Therapeutic Effect of Cryofiltration in a Patient With Glucocorticoid‐Resistant Ulcerative Colitis

Abstract:  Ulcerative colitis (UC) is characterized by chronic inflammation of the colon and its cause and pathogenesis have not been fully clarified. Although UC is treated with various drugs, including 5‐amino‐salycilate and glucocorticoids, some patients are resistant to them. It was recently reported that apheresis, such as leukocytapheresis and granulocytapheresis, improves intestinal inflammation in refractory cases of UC. On the other hand, cryofiltration, in which plasma apheresis is used to remove immunoglobulin and immune complexes, has been used for the treatment of autoimmune diseases. We herein report a case of glucocorticoid‐resistant UC successfully treated with cryofiltration. Interestingly, the level of interleukin‐10 (IL‐10) in the patients serum was markedly increased after eight sessions of cryofiltration. This suggests that cryofiltration suppresses intestinal inflammation, in part via up‐regulation of IL‐10.

Further accumulation of Cd in renal cellular membranes caused by administration of desferrioxamine to Cd-burdened rats

AbstractBackground. Desferrioxamine (DFO) a chelating agent, is used to treat metal toxicity caused by iron and aluminum in patients on hemodialysis. We hypothesized that DFO could also be used to treat cadrium-induced nephropathy. Animal experiments were therefore performed to explore whether DFO removed cadmium (Cd) from the kidneys of rats with a Cd burden. Methods. Rats received subcutaneous injections of Cd chloride (3 mg Cd/kg per day, days 0–7) followed by DFO (50 mg/kg per day, days 8–14). Levels of Cd were determined in liver, kidneys, and plasma. Enzymes assays and histopathological examination were performed in kidneys. Results. In liver, Cd injections elevated Cd levels; subsequent injections of DFO lowered the Cd levels compared with levels after injections of Cd alone. In kidneys, Cd injections increased levels of total Cd and Cd bound to cellular membranes (Mem-Cd), and decreased leucine aminopeptidase (LAP) activity (a marker of renal injury); subsequent injections of DFO elevated levels of total Cd and Mem-Cd, and lowered LAP activity compared with fundings after the injection of Cd alone. After the injections of Cd alone and DFO following Cd the renal levels of Cd were below the critical concentration required to cause renal injury, since no histopathological changes were observed in the kidney. Conclusion. DFO administration to Cd-burdened rats removed Cd from the liver, but led to accumulation of Cd in the kidneys, particularly in the cellular membranes. These results suggest that if DFO is given long-term to Cd-burdened patients, the Cd level in kidneys, particularly in renal cellular membranes, could reach concentrations that could cause manifest renal injury.

Problems of double-lumen catheter used for blood purification and their improvement

We discuss the problems of the double-lumen catheter (DLC) that we have used for blood purification and their improvement. The main problems are infection and insufficient blood flow during blood purification due to intraluminal thrombosis, the attachment phenomenon, and thrombosis of the individual problems, but all the problems have not yet been resolved. The development of a DLC that resolves almost all problems is desired in the near future.